The invention relates to a method and devices for far field microscopy and flow fluorometry for geometric distance measurements between object structures marked with fluorochromes, wherein the distances may be smaller than the half-intensity width of the principle maximum (=full width at half maximum of the intensity peak=FWHM) of the actual point spread function.
By employing highly specific markers, such e.g. DNA probes or protein probes, it is possible to mark practically any small (sub-)structures in biological (micro-)objects, in particular in cells, cell nuclei, cell organs or chromosomes (hereinafter also described as objects for abbreviation). Such markers may specifically represent structures in dimensions from several xcexcm (10xe2x88x926 m) to a few tens of nm (10xe2x88x929 m). In these markers normally reporter molecules are integrated, which have a high affinity with complex compounds, to which fluorochromes, but also colloidal microparticles (e.g. gold) are attached. Such fluorochromes/complexes can also be integrated directly into the markers. The available colour emission spectra of fluorochromes stretch from deep blue through green and red to the infrared range of the spectrum. Equally, fluorochromes can be used that do not differentiate in terms of excitation and/or fluorescence emission in their spectrum, but in which the life time of their fluorescence emission is used as a parameter for differentiation.
The latter have the advantage that focal shifts depending on the wavelength do not arise. Fluorochromes can also have a different emission spectrum and thus possess different spectral signatures, and yet be stimulated by the same photon energy, e.g. by means of multiple photon processes. It is also possible in this case to avoid wavelength-dependent focal shifts in the excitation between fluorochromes with different spectral signatures.
The above-named fluorochromes bound to specific (sub-) structures in biological micro-objects are referred to hereinafter as fluorescence markers. Fluorescence will be used below to encompass every photon interaction in which differences arise between a material""s stimulation spectrum and its emission spectrum that cannot be attributed to monochromatic absorption or dispersion. This also includes in particular multiple photon interactions in which the stimulation wavelengths may be greater than the emission wavelengths. Furthermore, the term fluorescence is also used here for the closely related phenomenon of luminescence, in particular phosphorescence. This includes in particular longer and medium term fluorescence life time, e.g. fluorescence life time in the range of up to several or many msec (milliseconds). The closely related processes of luminescence, phosphorescence and fluorescence will be treated herein as equally relevant to the invention. If the excitation spectrum and/or the emission spectrum and/or the fluorescence life time of two fluorescence markers agree, they have the same spectral signature based on the parameter in question. If they differ in one or more parameters relevant to the measurement, they have different spectral signatures.
A series of light microscopic measuring methods is used for detecting the fluorescence markers in extended biological objects and for the quantitative localisation relative to defined object points/object structures (distance and angle measurements). This is primarily a case of (a) epifluorescence microscopy, (b) confocal laser scanning microscopy, (c) laser scanning flow fluorometry, (d) the far field microscopy process of xe2x80x9cpoint-spread-function-engineeringxe2x80x9d and (e) standing wave field microscopy.
a) In the case of epifluorescence microscopy with a classical upright or inverse epifluorescence microscope, the biological object is illuminated by the same lens through which it is detected. The excitation light and the fluorescence emitted are discriminated by appropriate optical filters and conducted into different beam paths. The obtainable resolution, i.e. the smallest distance still measurable between two point-shaped object structures that are marked with fluorochromes with the same spectral signature, is given either by the Abbe criterion (=the maximum 0. order of the diffraction pattern of a point object is localised in the 1st minimum of the diffraction pattern of a second point object) or by the half-intensity width of the principle maximum of the actual point spread function. This depends on the wavelength, on the numerical aperture of the lens used and on the local refractive indices of the objects, of the embedding medium, of any cover slip used and of any immersion fluid applied. (In the case of a higher numerical aperture, its dimension may be smaller than the wavelength of the light used for stimulation).
b) In the case of confocal laser scanning microscopy, unlike epifluorescence microscopy, a laser is focused in the lens and the fluorescence is detected confocally. In order to create a three-dimensional image, the object is scanned in all three directions (x, y, z) with the focus point. As in the case of epifluorescence microscopy, the obtainable resolution is given by the half-intensity width of the principle maximum (FWHM)of the actual point spread function and depends on the wavelengths, on the numerical aperture of the lens used and on the local refractive indices of the objects, of the embedding medium, of any cover slip used and of any immersion fluid applied.
c) In the case of laser scanning flow fluorometry, the objects are conducted for example individually through an appropriate light distribution of the focus by a carrier fluid beam that is free or situated or in an optical cuvette (while in the case of epifluorescence microscopy and confocal laser scanning microscopy, the objects are predisposed in a fixed position on object carriers, i.e. object slides, capillaries, chambers, fluids etc.). The light distribution is normally slit-shaped, i.e. the object is scanned with reference to an axis. The obtainable resolution is determined by the width of the focus of the laser beam used and/or suitably selected detection scans, wherein the variability in the object trajectory (=laminar, usually central xe2x80x9cfluid filamentsxe2x80x9d that carry the object) allows for the focal depth and thus also the minimal focal width, regardless of the carrier medium and method. The advantage of flow fluorometric methods is usually found in the relatively higher detection rate, compared to epifluorescence microscopy and confocal laser scanning microscopy, which can reach several thousand objects per second. The focal width complies with the full half-intensity width of the principle maximum of the actual point spread function of the slit-scan optics in the conditions used.
d) In the case of the far field microscopy technique of xe2x80x9cpoint-spread-function-engineeringxe2x80x9d, the point spread function is reduced in width optically. This may be achieved by means of coherently overlapping two or more point spread functions (e.g. 4Pi microscopy) or by means of extinguishing the fluorescence of fluorochromes that are situated in the peripheral area of the central point spread function maximum in question (e.g. STED microscopy, ground depletion microscopy). As the resolution of a microscope is given by the full half-intensity width of the principle maximum of the actual point spread function, the half-intensity width is thus reduced and the resolution improved.
e) In the case of standing wave field microscopy according to U.S. Pat. No. 4,621,911, luminescent preparations are illuminated with a standing wave field in an optical microscope (standing wave field fluorescent microscopy, SWFM). The preparations are set in a zone of equidistant wave fronts and stimulated to fluorescence or phosphorescence. The space between the wave fronts and their phases can be varied to generate patterns. The three-dimensional distribution of fluorescent or luminescent object points can be reconstructed from individual optical sections by means of computer image processing.
The planar wave fronts are generated by coherently overlapping two laser beams at a defined angle to the optical axis of the microscope system, whereby the angle determines the distance between the wave frontsxe2x80x94with given wavelengths and refraction indices. Instead of two intersecting laser beams, the standing wave field can also be generated by making a laser beam interfere with itself at a certain angle after suitable reflection. In microscope construction in these cases, the wave fronts are set perpendicularly to the optical axis of the detecting lens. The fluorescence or luminescence is either spectrally discriminated by means of appropriate optical filters and conducted into different beam paths, as in the epifluorescence microscope, or detected confocally. As in the case of epifluorescence microscopy and confocal laser scanning microscopy, the obtainable resolution is given by the full half-intensity width of the principle maximum of the actual point spread function and depends on the given wavelength, on the numerical aperture of the lens used and on the local refractive indices of the objects, of the embedding medium, of any cover slip used and of any immersion fluid applied.
Laterally, the system has a resolution akin to a conventional epifluorescence microscope or a confocal laser scanning microscope; axially, on the other hand, a depth discrimination and thus a considerably better resolution is achieved.
1) As the actual point spread functions are strongly influenced by the local refraction index and absorption in the object, in the object""s embedding medium and in the immersion (including any cover slips present), the measurement of distances between object structures depends on the actual point spread function-i.e. the one given locally in the marked object point. This generally differs clearly from calculated point spread functions of the microscope used. The technically optimised marginal conditions of measured point spread functions also generally differ from the actual point spread functions obtainable in biological objects under practical routine laboratory conditions. As these actual point spread functions are mostly not available, distance measurements for calibrating purposes are usually made by referring to ideal, calculated results or to calibration measurements that are made under standard conditions, such as e.g. reflection methods. Both methods are detrimental to accuracy in three-dimensional distance measurement in biological micro-objects. As a result, there is considerable uncertainty in the determination of the real spatial distance between the object structures; in the case of biological objects, quantitative size estimates contain uncertainties of up to several micrometers. There is only a limited possibility to correct this error with the methods used hitherto, i.e. only under standard conditions, whose actual achievement/compliance in the biological object cannot be controlled or guaranteed accurately, however.
Two object structures with the same spectral signature can then only be separated if the distance between them is at least a half-intensity width of the principle maximum of the actual point spread function.
2) All the ar field methods described above suffer from the problem that the half width of the principle maximum of the point spread function and thus the limits of resolution depend on the relative position in space. Thus, in the case of epifluorescence microscopy or confocal laser scanning microscopy, for example, the point spread function is narrower laterally (perpendicular to the optical axis) than axially (in the direction of the optical axis). In the case of static microscopy methods, this disadvantage can be overcome with the aid of so-called micro-axial tomography. In this method, the (biological) objects are set in capillaries or on glass fibres and rotated around an axis under defined conditions in the microscope, which is normally perpendicular to the microscope""s optical axis. In this case, distance measurements are made in the direction with the narrowest half-intensity width of the actual point spread function. This method can hardly be applied, however, in the case of flow fluorometry.
3) In the case of xe2x80x9cmono-dimensionalxe2x80x9d standing wave field microscopy (SWFM), the periodical wave field in the case of epifluorescent detection in connection with optical sectioning leads to ambiguity in the image of the object structures greater than xcex/2 n (xcex=wavelength of the excitation, n=actual refraction index). This ambiguity primarily makes it difficult to make any effective use of the improvement in resolution achieved by means of the interference pattern.
4) According to the state of the art, high precision distance measurements with light microscopy far field methods can only be made as far as the range of dimensions of a hundred nanometers. For measurements in the range of distances and accuracy of the order of 10 nm, the methods of electron microscopy, scanning tunnel microscopy, atomic force microscopy and biological and optical near-field microscopy are used. Nevertheless, these are surface-oriented and not volume-oriented methods-unlike the optical far field method; in other words, they are in principle useful only for structural investigations and distance measurements on surfaces and in thin layers. In any event, information about the position of objects or object structures in three dimensions can be obtained using mechanically prepared cut series and evaluating measurements in individual image sections. Three-dimensional measurements in intact or even vital biological micro-objects, such as three-dimensional (conserved) cells, cell nuclei or cellular organs, are not possible.
The task of the invention is to prepare a method for far field light microscopy and a device for executing this method, with which it is possible to make highly accurate distance measurements between object structures whose reciprocal distance is smaller than the resolution capacity of the far field microscope in question, i.e. that are separated by less than the half-intensity width of the maximum of the actual point spread function, regardless of the position of the object structures in question in the three-dimensional space.
One solution to this task consists of preparing a method of the kind described above that is a calibrating process for fluorescence far field microscopy and includes the following process steps:
Before, during or after the preparation of the object in question on or in an object carrier, in particular object slides, fibres/capillaries or fluids, the structures to be investigated or localised (measurement structures) are labelled with fluorescent dyes with different and/or identical spectral signatures, i.e. such structures (measurement structures) to be localised as are located in each others"" immediate vicinities, i.e. within the half-intensity width of the principle maximum of their actual point spread function, are labelled with fluorescent dyes with different spectral signatures, while such measurement structures as are located at distances greater than that of the half-intensity width of the principle maximum of the actual point spread function are labelled with fluorescent dyes with different or equal spectral signatures. Two measurement structures to be localised can thus always be labelled with the same spectral signature, if for example they can be identified clearly by means of their relative positions or other criteria.
calibration targets of defined sizes and spatial arrays are labelled with the same fluorescent dyes,
the fluorescent calibration targets are prepared, either together with the objects or separately on or in an object carrier (object slides, fibres/capillaries, fluids or the like).
objects (of investigation) and calibration targets are investigated microscopically or flow fluorometrically in corresponding conditions, simultaneously or one after another.
pairs of defined calibration targets with different spectral signatures are measured in consideration of the wavelength-dependent imaging and localisation behaviour of the optical system in question (microscope or flow fluorometer); the values thus measuredxe2x80x94the actual valuesxe2x80x94are compared to the previously known real distance valuesxe2x80x94the nominal values (i.e. the nominal localisation calculated on the basis of geometry), and the difference between the actual values and the nominal values, i.e. the calibration value, is used to correct the offset caused by the optical system in the detection of different emission loci, in particular in the measurement structures.
In other words, the distance measurement between the object (sub-) structuresxe2x80x94hereinafter also described as measurement structuresxe2x80x94marked (according to the distances between them) with different or identical spectral signatures is made using the high precision localisation of independent (calibration) targets with a conforming spectral signature with a known size and spatial array, in consideration of the wavelength-dependent imaging and localisation behaviour of the optical system in question, whereby the calibration measurement between the (calibration) targets and the measurement in the biological objects takes place in the same system and marginal conditions. These calibration targets have the same or a higher multi-spectrality as or than the (object) structures to be measured. They can be arrayed directly in the biological objects or as a separate preparation on an object carrier (object slides, fibres/capillaries, fluids or the like) or be part of an object carrier.
Two or more fluorescent measurement structures in intact, three-dimensional biological objects, whose distance and extension is less than the half-intensity width of the principle maximum of their actual point spread function, can be discriminated on the basis of their different spectral signature (fluorescence absorption wavelengths and/or fluorescence emission wavelengths and/or fluorescence emission life time), i.e. their distances can be determined.
The distance measurement can be reduced to the localisation of the individual measurement structures and canxe2x80x94now also in optical far field microscopy or flow fluorometryxe2x80x94by carried out with a considerably higher accuracy than the half-intensity width of the maximum of the point spread function. The localisation of the barycentre of the measurement structures in question is adapted to the maximum intensity of their fluorescence signals. In other words, the barycentre of the signal and thus the location of the measurement structure are determined from the measured (diffraction limited) signal (=intensity curve) of a fluorescence point (=fluorescence measurement structure)-in consideration of the overall information from the minor and major maxima. In the case of an error-free optical system and consequently ideal symmetry of the measured intensity distribution (=course of the intensity curve), the barycentre of the intensity curve co-localises within the localisation accuracy with the main maximum (=maximum 0. order of the diffraction pattern) of the measured intensity distribution.
The method in accordance with the present invention enables optical far field region microscopy or scanning flow fluorometry to be used to measure distances in biological micro-objects, whereby the distances to be determined may be smaller than the half-intensity width of the principle maximum of the actual point spread function in the object. As the information content of the distance determination carried out in accordance with the invention complies with a distance measurement made with higher resolution, it is also possible to speak summarily in terms of resolution equivalent. The measurements of such small distances in biological micro-objects is of great significance for example for scientific questions in biology and medicine, but also for certain aspects of clinical research and diagnostics of preparations.
Multispectral calibration allows in situ measurements to be made of concrete biological objects via the system""s image behaviour. If fluorescence life time is used as the only type of parameter and/or if the fluorochromes are excited with the same photon energy/ies, the in situ correction of the chromatic shift in the object plane is no longer necessary. For high resolution far field microscope types, such as e.g. the standing wave field microscope, and when fluorescence markers according to the invention are used, the invention enables three-dimensional geometric distance measurements to be made in biological objects right down to molecular accuracy (i.e. resolution equivalent better than 10 nm).
Unlike electron microscopy or optical or non-optical near field microscopy, the three-dimensional structure of the object to be investigated remains intact, as there is no need for mechanical sectioning. 3D distance measurements with a range smaller than the half-intensity width of the maximum of the actual point spread function can thus be undertaken in three-dimensionally conserved micro-objects. In particular, the method makes it possible to undertake three-dimensional distance measurements also in vital conditions of the biological object. Compared to the methods of point spread function engineering known to the state of the art, a considerable advantage in the invention consists of the fact that already existing systems for quantitative fluorescence microscopy can also be used as a basis for the increase according to the invention of the resolution equivalent.
Both one and two-dimensional scanning and also non-scanning electronic or opto-electronic detector systems are suitable for detecting the fluorescence emission.
One application of the method according to the invention is particularly useful for multispectral precision distance measurements in biological micro-objects for the absolute and relative localisation and distance measurement of fluorescent measurement structures with any spectral signature.